丙泊酚对大鼠海马神经元缺氧/复氧损伤中线粒体分裂及其超微结构的影响

目的 探讨丙泊酚在大鼠海马神经元缺氧/复氧损伤模型中对线粒体分裂及其超微结构的影响. 方法 培养原代海马神经元细胞至第8天,氧糖剥夺法建立海马神经元缺氧/复氧模型,按照随机数字表法分为6组(每组6瓶):空白对照组(C组),细胞未给予任何处理;赋形剂组(V组),赋形剂[二甲基亚砜(dimethyl sulfoxide,DMSO),终浓度为0.01％]加入细胞培养基;缺血/再灌注(ischemia/reperfusion,I/R)组(I/R组);I/R+丙泊酚1μmol/L组(P1组)、I/R+丙泊酚10μmol/L组(P10组)、I/R+丙泊酚50 μmol/L组(P5o组),在细胞缺氧/复氧期间分别加入丙泊酚1、10、50 μmol/L.缺氧6h,复氧20 h后,用透射电子显微镜观察线粒体超微结构,激光共聚焦显微镜检测神经元细胞线粒体荧光强度及Drp1与Fis1蛋白的共定位程度,用Western blot检测线粒体分裂相关蛋白Drp1 、Fis1的表达. 结果 与C组比较,I/R组线粒体超微结构破坏明显、线粒体荧光强度(0.079±0.032)明显增高(P＜0.05),蛋白Drp1 (0.756±0.082)与Fis1 (1.164±0.070)的表达及共定位程度(0.815±0.048)明显升高(P＜0.05);与I/R组比较,P1组、P10组、P50组线粒体超微结构破坏减轻、线粒体荧光强度(0.065±0.010、0.056±0.011、0.070±0.024)明显减弱(P＜0.05),蛋白Drp1(0.627±0.005、0.322±0.009、0.696±0.007)与Fis1(0.773±0.012、0.670±0.022、0.796±0.016)的表达及共定位程度(0.649±0.015、0.627±0.008、0.702±0.029)明显降低(P＜0.05). 结论 丙泊酚1、10、50 μmol/L可以抑制体外大鼠海马神经元中线粒体分裂相关蛋白Drp1与Fis1的表达及两者的结合,从而抑制线粒体分裂.

The effect of propofol on mitochondrial ultrastructure and mitochondrial fission following oxygen/reoxygenation injury in hippocampal neurons of rats

Objective To investigate the effect of propofol on mitochondrial ultrastructure and mitochondrial fission during oxygen-glucose deprivation and reperfusion injury in hippocampal neurons of rats.Methods Cultured primary hippocampal cells were subjected to oxygen-glucose deprivation for 6 h,followed by 20 h of reperfusion and then were randomly divided into 6 groups (n=6):control group (group C),vehicle group (group V),group ischemia/reperfusion(I/R),I/R+propofol(P1,P10,P5o) treatment groups,propofol 1,10,50 μmol/L were added during oxygen-glucose deprivation and reperfusion period.Mitochondrial ultrastructure (using atransmission electron microscop),fluorescence intensity of mitochondria and quantitative colocalization of Drp 1 and Fis 1(using a laser scanning confocal microscope),expression of Drpt and Fis1 (by Western blot) were measured.Results Compared with group C,mitochondrial ultrastructure were destroyed and fluorescence intensity of ruitochondria (0.079±0.032) and quantitative colocalization of Drp1 and Fis1(0.815±0.048),the expression of Drp1(0.756±0.082) and Fis1 (1.164±0.070) were increased in other groups (P＜0.05).Compared with group I/R,mitochondrial ultrastructure were improve(P＜0.05) and fluorescence intensity of mitochondria(0.065±0.010,0.056±0.011,0.070±0.024)(P＜0.05) and quantitative colocalization of Drp1 and Fis1 (0.649±0.015,0.627±0.008,0.702±0.029),the expression of Drp1(0.627±0.005,0.322±0.009,0.696±0.007) and Fis1(0.773±0.012,0.670±0.022,0.796±0.016) were decreased in P1,P10 and P50 groups (P＜0.05).Conclusions Propofol 1,10,50 μnol/L could inhibit I/R-induced mitochondrial fission by suppressing the expression and the binding of Drp1 and Fis1,of which 10 μmol/L was the optimal dose in hippocampal neurons of rats.